發(fā)布時(shí)間：2018-02-23 17:37

  本文關(guān)鍵詞： RPL10 RPL10L X染色體起源的逆轉(zhuǎn)座基因 減數(shù)分裂性染色體沉默 補(bǔ)償假說　出處：《中國(guó)科學(xué)技術(shù)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：哺乳動(dòng)物的性染色體是從祖先的常染色體進(jìn)化而來,在整個(gè)進(jìn)化過程中經(jīng)歷了復(fù)雜的變化。例如,X染色體上的基因數(shù)量在真獸亞綱哺乳動(dòng)物中還有兩次爆發(fā)性的增長(zhǎng)�；虻霓D(zhuǎn)移和重新分配賦予了 X染色體的特殊功能。大量研究表明起源于X染色體的逆轉(zhuǎn)座基因在基因組中所占的比例遠(yuǎn)遠(yuǎn)高于起源于常染色體的比例,這類基因往往定位于常染色體并且具有睪丸特異表達(dá)的特點(diǎn)�？茖W(xué)家們也提出了一些基于進(jìn)化選擇的假說用于解釋這種現(xiàn)象,包括性別對(duì)抗驅(qū)動(dòng)X染色體失活假說以及減數(shù)分裂性染色體失活補(bǔ)償假說等。然而,這些假說都缺乏直接的實(shí)驗(yàn)證據(jù)。盡管有幾個(gè)X染色體起源的逆轉(zhuǎn)座基因已被證實(shí)對(duì)精子發(fā)生至關(guān)重要,但這些基因與其前提基因的進(jìn)化、表達(dá)及功能相關(guān)性仍未見報(bào)道。RPL10L是一個(gè)位于常染色體并在睪丸中特異表達(dá)的逆轉(zhuǎn)座基因,它的前體是X染色體上一個(gè)核糖體蛋白編碼基因RPL10,它的進(jìn)化發(fā)生在真獸亞綱和后獸亞綱分支后不久的真獸亞綱哺乳動(dòng)物中。我們選擇RPL10L和RPL10作為模型,詳細(xì)研究它們的表達(dá)模式、分子功能及作用方式,以期深入了解X染色體來源的逆轉(zhuǎn)座基因存在的意義及其進(jìn)化機(jī)制。首先,我們利用CRISPR/Cas9的方法制作了Rpl10l敲除小鼠,并發(fā)現(xiàn)雄性個(gè)體的精子發(fā)生障礙并且完全不育,具體表現(xiàn)為絕大多數(shù)精母細(xì)胞發(fā)育停滯在減數(shù)第一次分裂前期向中期的轉(zhuǎn)換,沒有減數(shù)分裂后的生殖細(xì)胞產(chǎn)生。其次,RPL10L蛋白特異地參與睪丸細(xì)胞中的核糖體組裝,缺失它的粗線期及雙線期精母細(xì)胞中的核糖體生成減弱并且大量蛋白的含量發(fā)生變化。再次,Rpl10在雌雄小鼠不同組織以及精子發(fā)生的精原細(xì)胞、前細(xì)線期精母細(xì)胞、細(xì)線期及偶線期精母細(xì)胞中廣泛表達(dá),但在粗線期及雙線期精母細(xì)胞和圓形精細(xì)胞中的表達(dá)量急劇下降;有趣的是,Rpl10l在精子發(fā)生的粗線期及雙線期精母細(xì)胞和圓形精細(xì)胞中特異地高表達(dá)。最后,RPL10敲低的細(xì)胞中核糖體生成受阻并伴隨細(xì)胞周期停滯,RPL10敲除的細(xì)胞無法增殖和存活;而外源導(dǎo)入RPL1OL表達(dá)載體能夠補(bǔ)救RRL10敲除細(xì)胞的增殖和存活;更重要的是,Rpl10l啟動(dòng)子驅(qū)動(dòng)的Rpl10轉(zhuǎn)基因能夠重建Rpl10l敲除小鼠的精子發(fā)生和生育力。綜上所述,我們的研究證實(shí)Rpl10l能夠補(bǔ)償MSCI導(dǎo)致的Rpl10轉(zhuǎn)錄沉默以保證正常的精子發(fā)生,為基于MSCI的逆轉(zhuǎn)座基因補(bǔ)償假說提供了直接的實(shí)驗(yàn)證據(jù),也為X染色體起源的逆轉(zhuǎn)座基因的進(jìn)化機(jī)制以及其在雄性生殖過程中的作用提供了新的理論依據(jù)和見解。
[Abstract]:The sexual chromosomes of mammals evolved from the autosomes of their ancestors. During the course of evolution, there have been complex changes. For example, the number of genes on the X chromosome has increased twice in mammals of the subclass Encalypta. The transfer and redistribution of genes have given the X chromosome special. Specific function. A large number of studies have shown that the proportion of genes originating in the X chromosome in the genome is much higher than that in the autosomal genome. Such genes are often located on autosomes and have testicle-specific characteristics. Scientists have also come up with hypothesis based on evolutionary selection to explain the phenomenon. These include the X chromosome inactivation hypothesis driven by gender antagonism and the meiotic chromosome inactivation compensation hypothesis. However, These hypotheses lack direct experimental evidence. Although several reverse locus genes from the origin of X chromosomes have been proven to be essential for spermatogenesis, these genes and their underlying genes evolved. Expression and functional correlation have not been reported. RPL10L is a reverse locus gene located on autosomal and specifically expressed in testis. Its precursor is RPL10, a ribosomal protein coding gene on the X chromosome, which evolved in mammals of the order Euphorbia and the suborder of Postzoa. We chose RPL10L and RPL10 as models. Their expression patterns, molecular functions and modes of action were studied in detail in order to understand the significance of the existence of the reverse locus gene from the X chromosome and its evolutionary mechanism. Firstly, Rpl10l knockout mice were made by using CRISPR/Cas9 method. It was also found that the spermatogenesis of male individuals was impaired and completely sterile, which was manifested by the transformation of the majority of spermatocytes from the early to the middle stage of the first meiosis. No meiotic germ cells were produced. Secondly, RPL10L protein was specifically involved in ribosomal assembly in testicular cells. The ribosomal production of spermatocytes in the absence of Rpl10 decreased and the content of a large number of proteins changed in the spermatocytes of the two stages. Rpl10 was found again in spermatogonia and spermatogonia in different tissues of female and male mice, and spermatogonia in the spermatogonium of pretenline stage. The expression of spermatocytes was widely expressed in the fine and even-line stages, but decreased sharply in the spermatocytes and round spermatocytes in the coarse and double-line stages. It is interesting to note that Rpl10l is highly expressed in spermatocytes and round spermatocytes in the coarse-line and double-line stages of spermatogenesis. At last, ribosome production in the cells with low RPL10 knockout and RPL10 knockout with cell cycle arrest can not proliferate and survive. The exogenous RPL1OL expression vector can remedy the proliferation and survival of RRL10 knockout cells, and more importantly, the Rpl10 gene driven by Rpl10l promoter can reconstruct the spermatogenesis and fertility of Rpl10l knockout mice. Our study confirmed that Rpl10l can compensate for Rpl10 transcriptional silencing induced by MSCI to ensure normal spermatogenesis, providing direct experimental evidence for the MSCI gene compensation hypothesis. It also provides a new theoretical basis and understanding for the evolutionary mechanism of the reverse locus gene of the origin of X chromosome and its role in male reproduction.
【學(xué)位授予單位】：中國(guó)科學(xué)技術(shù)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q132.1
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本文編號(hào)：1527008